Mavrangelos Chris, Wijenayaka Asiri, Sales Kurt J, Hughes Patrick A
Agilex Biolabs, Adelaide, South Australia, Australia.
Adelaide Medical School, University of Adelaide, Adelaide, South Australia, Australia.
Immunohorizons. 2025 Jan 27;9(3). doi: 10.1093/immhor/vlae012.
Enzyme-linked immunosorbent spot analysis is frequently used to investigate immune responsiveness during clinical trials. However, ELISpot classically utilizes peripheral blood mononuclear cell isolates from whole blood, requiring relatively high blood draw volumes and removing both granulocytes and bound drug. Here, we describe a novel protocol whereby CD45 cells are magnetically isolated from human whole blood and co-incubated with serum isolated from the same subject. Infliximab is a well characterized anti-tumor necrosis factor α (TNF-α) antibody in clinical use since the late 1990s. We demonstrated that TNF-α inhibition by infliximab in spiked whole blood is lost on peripheral blood mononuclear cell isolation but remains in serum, and that combining serum from infliximab spiked whole blood with magnetically isolated CD45 immune cells inhibited PMA/ionomycin-stimulated TNF-α secretion. This novel protocol has important implications for enzyme-linked immunosorbent spot analysis in clinical trials in which blood volume is limited, and keeping drug responses intact provides critical information.
酶联免疫斑点分析常用于临床试验中研究免疫反应性。然而，传统的酶联免疫斑点法利用全血中的外周血单个核细胞分离物，需要相对大量的采血，并且会去除粒细胞和结合的药物。在此，我们描述了一种新方案，即从人全血中磁性分离CD45细胞，并与从同一受试者分离的血清共同孵育。英夫利昔单抗是一种自20世纪90年代末以来在临床中使用的、特征明确的抗肿瘤坏死因子α（TNF-α）抗体。我们证明，英夫利昔单抗在加标的全血中对TNF-α的抑制作用在外周血单个核细胞分离时丧失，但在血清中仍然存在，并且将来自英夫利昔单抗加标全血的血清与磁性分离的CD45免疫细胞相结合可抑制佛波酯/离子霉素刺激的TNF-α分泌。这种新方案对于血容量有限的临床试验中的酶联免疫斑点分析具有重要意义，并且保持药物反应完整可提供关键信息。
